The human immunodeficiency virus encodes a highly conserved transcriptional trans-activator Tat, which is expressed early in the viral life cycle and is essential for viral replication and progression to disease (Cullen et al., Cell 73:417-20 (1993); Jones et al., Annu. Rev. Biochem. 63:717-743 (1994)). Tat binds to the trans-activation response (TAR) RNA stem-loop located from positions +1 to +60 in the viral 5' long terminal repeat (LTR). Interactions between Tat and TAR are absolutely required for the increased processivity of RNA polymerase II (pol II) and the production of full-length viral transcripts (Kao et al., Nature (London) 330:489-493 (1987); Laspia et al., Cell 59:283-292 (1989); Marciniak et al., EMBO J. 10:4189-4196 (1991); Kato et al., Genes Dev. 6:655-666 (1992)). Tat is unique because it is the only eukaryotic transcription factor known to function via RNA (Madore et al., Virology 206:1150-1154 (1995)). Although the mechanism by which Tat increases transcription elongation rates is unknown, common regulatory themes must exist between viral and cellular genes since Tat can relieve pol II pausing when artificially targeted to the c-myc promoter (Wright et al., J. Mol. Biol. 243:568-573 (1994)).
Tat can be divided into two functional domains. The activation domain contains 48 N-terminal amino acids and interacts with the cellular transcriptional machinery. A 10 amino acid basic domain is required for the binding of Tat to TAR (Jones et al., Annu. Rev. Biochem. 63:717-743 (1994)). Cellular proteins are clearly required for the function of Tat (Carroll et al., J. Virol. 66:2000-2007 (1992); Madore et al., J. Virol. 67:3703-3711 (1993)). At least one protein, encoded by the human chromosome 12, is required for the efficient binding of Tat to TAR (Hart et al., Science 246:488-491 (1989); Alonso et al., J. Virol. 66:4617-4621 (1992)). Numerous other proteins have been postulated to interact with the activation domain of Tat. These include general transcription factors (GTFs) such as the core pol II (Mavarikal et al., Proc. Natl. Acad. Sci. USA 93:2089-2094 (1996)), the TATA box-binding protein (TBP) (Kashanchi et al., Nature (London) 367:295-299 (1994); Veschambre et al., J. Mol. Biol. 250:169-180 (1995)), the TBP-associated factor TAF.sub.II55 (Chiang et al., Nature (London) 267:531-536 (1995)) and TFIIH (Blau et al., Mol. Cell. Biol. 16:2044-2055 (1996); Parada et al., Nature (London) 384:375-378 (1996)). Upstream DNA bound activators such as Sp1 have also been identified as possible co-activators of Tat (Jeang et al., J. Virol. 67:6224-6233 (1993)). In addition, a wide variety of other proteins that interact with Tat but whose role in transcription is somewhat unclear have also been identified (Nelbock et al., Science 248:1650-1653 (1990); Desai et al., Proc. Natl. Acad. Sci. USA 88:8875-8879 (1991); Zhou et al., Science 274:605-610 (1996)).
Recently, Tat has been demonstrated to interact with the pol II holoenzyme (Cujec et al., Mol. Cell. Biol. 17:1817-1823 (1997)). This large mega-dalton complex consists of core pol II, a subset of general transcription factors (TFIIE, TFIIF, TFIIH), human SRBs (suppressors of mutations in polymerase B), which confer the ability of the pol II holoenzyme to respond to activators, and proteins involved in chromatin remodeling (SWI/SNF) and nucleotide repair (Kim et al., Cell 77:599-608 (1994); Ossipow et al., Cell 83:137-146 (1995); Chao et al., Nature (London) 380:82-85 (1996); Maldonado et al., Curr. Opin. Cell Biol. 7:352-361 (1995); Wilson et al., Cell 84:235-244 (1996)). One component of the pol II holoenzyme, TFIIH, contains nine polypeptides (ERCC3, ERCC2, p62, p54, p44, CDK7 (MO15), cyclin H, MAT 1, and p34) (Drapkin et al., Trends Biochem. Sci. 19:504-508 (1994); Hoeijmakers et al., Curr. Opin. Genet. Dev. 6:26-33 (1996)).
TFIIH also contains a kinase activity that can phosphorylate the C-terminal domain (CTD) of core pol II (Feaver et al., Cell 67:1223-1230 (1991); Lu et al., Nature 358:641-645 (1992)). The kinase activity resides in the cyclin-dependent kinase 7 (CDK7) polypeptide (Feaver et al., Cell 79:1103-1109 (1994); Roy et al., Cell 79:1093-1101 (1994); Serizawa et al., Nature (London) 374:280-282 (1995); Shiekhattar et al., Nature (London) 374:283-287 (1995)). In association with cyclin H, CDK7 forms the CDK-activating kinase (CAK) complex, which phosphorylates cyclin-dependent kinases (CDKs) involved in the regulation of the cell cycle. Association of MAT 1 with the CAK dimer stabilizes the complex and allows for the activation of CAK independent of the phosphorylation of CDK7 on the threonine at position 170 (Fisher et al., Cell 78:713-724 (1994); Fisher et al., Cell 83:47-57 (1995)). Moreover, the CAK trimer is much more efficient at phosphorylating the CTD than the CAK dimer (Rossignol et al., EMBO J. 16:1628-1637 (1997); Yankulov et al., EMBO J. 16:1638-1646 (1997)). The tripartite CAK can exist in three distinct complexes in cells. The majority is present as free CAK. However, CAK can also exist as a CAK-ERCC2 complex as well as in association with the core TFIIH (ERCC3, p62, p54, p44 and p34) (Drapkin et al., Proc. Natl. Acad. Sci. USA 93:6488-6493 (1996); Reardon et al., Proc. Natl. Acad. Sci. USA 93:6482-6487 (1996)). The association of CAK with TFIIH confers kinase activity to TFIIH and renders it transcriptionally competent. Interestingly, the yeast homologue of CDK7, Kin28 is found only in a complex with TFIIH and is devoid of CAK activity (Cismowski et al., Mol. Cell. Biol. 15:2983-2992 (1995)). Instead, CAK activity resides in a novel protein called Civ1 or CAK1p (Kladis et al., Cell 86:553-564 (1996); Thuret et al., Cell 86:565-576 (1996)).
The eukaryotic pol II is unique among polymerases in that it contains multiple heptapeptide repeats of the sequence (YSPTSPS) at the C-terminal end of the protein, which comprise the CTD (Dahmus, Prog. Nucleic Acid Res. Mol. Biol. 48:143-179 (1994); Dahmus, Biochim. Biophys. Acta. 1261:171-182 (1995)). A large number of kinases capable of phosphorylating the CTD in vitro have been identified. However, the functional relevance of these kinases remains unclear. To date, CDK7/cyclin H (TFIIH) and CDK8/cyclin C (human homologues of the yeast SRB10/SRB11) are the major kinases associated with transcription factors that can phosphorylate the CTD (Liao et al., Nature (London) 374:193-196 (1995); Serizawa et al., Nature (London) 374:280-282 (1995); Shiekhattar et al., Nature (London) 374:283-287 (1995)).
Pol II enters into the assembling transcription complex with its CTD unphosphorylated (IIA form). However, the CTD of elongating polymerases is highly phosphorylated (IIO form), primarily on its serine and threonine residues (Uchiumi et al., J Biol Chem 265:89-95 (1990); Laybourn et al., J. Biol. Chem. 265:13165-13173 (1990)). This observation led to the suggestion that the phosphorylation of the CTD is important for promoter clearance and for the processivity of pol II. Numerous additional observations support this contention: 1) the CTD of polymerases paused on the Drosophila hsp 70 promoter prior to heat shock activation are hypophosphorylated (IIA) while those of actively elongating polymerases are hyperphosphorylated (IIO) (O'Brien et al., Nature (London) 370:75-77 (1994)), 2) inhibitors of CTD kinases inhibit promoter clearance and elongation of pol II in vitro (Yankulov et al., J. Biol. Chem. 270:23922-23925 (1995); Yankulov et al., Mol. Cell. Biol. 16:3291-3299 (1996)), 3) the kinase activity of TFIIH is required for the clearance of the DHFR promoter but not for the initiation of its transcription (Akoulitchev et al., Nature (London) 377:557-560 (1995)), and 4) mutations in the yeast pol II CTD, the yeast homologue of CDK7 (Kin 28), or SRB2, a subunit of the pol II holoenzyme, each inhibit the processivity of pol II in vivo (Akhtar et al., EMBO J. 15:4654-4664 (1996)). The identification of CTD-binding proteins with homology to serine/arginine-rich (SR) proteins suggest that the phosphorylation of the CTD might also provide a mechanism for coupling transcription and pre-mRNA processing (Yuryev et al., Proc. Natl. Acad. Sci. USA 93:6975-6980 (1996)).
Recently, it has been demonstrated that the CTD is absolutely required for the production of long transcripts from the HIV LTR in vitro and in vivo (Chun et al., J. Biol. Chem. 271:27888-27894 (1996); Okamoto et al., Proc. Natl. Acad. Sci. USA 93:11575-11579 (1996); Parada et al., Nature (London) 384:375-378 (1996); Yang et al., J. Virol. 70:4576-4584 (1996)). In contrast, basal transcription and the production of short transcripts from the HIV LTR is independent of the CTD. Because TFIIH kinase activity is involved with CTD phosphorylation and Tat trans-activation, there is a need to identify the specific interactions between TAT and TFIIH, in order to develop inhibitors of Tat trans-activation of HIV transcription. Such inhibitors are needed to develop HIV therapeutics.